Prunella vulgaris L. active components and their hypoglycemic and antinociceptive effects in alloxan-induced diabetic mice.

Prunella vulgaris L. (Lamiaceae) (PV) is a herbaceous plant traditionally utilized in management of diabetes and it has immunomodulatory activity. In this study, acute and subchronic antidiabetic, in-vivo antioxidant and antinociceptive effects of PV were evaluated in alloxan-induced type 1 diabetes (T1D) in a mouse model. Bio-guided fractionation, isolation, RP-HPLC, and 1H and 13C NMR identification of the active components responsible for PV effects were determined. RP-HPLC analysis showed that PV contained rosmarinic acid (RA) 4.5%, caffeic acid (CA) 9.8% and p-coumaric acid (pCA) 11.6%. Bio-guided fractionation showed that PV most active fraction was rich in caffeic acid, hence named, caffeic acid-rich fraction (CARF). RP-HPLC, and 1H and 13C NMR experiments showed that CARF contained CA (93.4%) and RA (6.6%). CARF reduced blood glucose levels and improved in-vivo oxidative-stress. It also inhibited the carbohydrate-hydrolyzing enzymes (alpha-amylase and alpha-glucosidase) and reduced HbA1c levels more significantly (p≤0.05) than that of PV and equivalent amounts of CA or RA. For longer times, CARF had significantly (p≤0.05) increased serum-insulin, ameliorated thermal hyperalgesia and tactile allodynia more significantly (p≤0.05) than the effects of PV and equivalent amounts of CA or RA. Moreover, the tested compounds showed potential restoration of the lipid peroxide levels. Consequently, CARF and PV observed increase in serum-insulin, attenuation of alpha-amylase and alpha-glucosidase, and their antioxidant potentials might be responsible for their antidiabetogenic and antinociceptive properties. In conclusion, CARF isolated from PV could be a potential therapeutic agent to ameliorate T1D and related complications.

Synthesis of imidazo[1,2-a]pyridines in a sequential one-pot Groebke-Blackburn modification using 2-aminopyridines, aldehydes and amines.

Herein we present a novel synthetic procedure for the synthesis of imidazo[1,2-a]pyridines in a modified Groebke-Blackburn fashion. In a sequential three-step one-pot protocol the commercially hardly available isocyanide-component is formed in situ using standard reagents. Cyclization to the desired products can be afforded in the same reaction mixture. The absent need of isolation of the isocyanide in this protocol eases its handling considerably and workup is only needed to finally furnish the imidazo[1,2-a]pyridines via coloumn chromatography. This protocol is a convenient way to more diverse libraries of imidazo[1,2-a]pyridines extending the functionality of the Groebke-Blackburn synthesis.

Detection of pyrrolizidine alkaloids in German licensed herbal medicinal teas.

BACKGROUND: Because of the hepatotoxic, mutagenic, and cancerogenic effects of pyrrolizidine alkaloids (PAs) the German Federal Institute for Risk Assessment (BfR) recommends not to exceed a daily PA intake of 0.007 µg/kg body weight (0.42 µg/60 kg adult). In a recent study conducted by the BfR, up to 5647 µg PA/kg dried herbal material were detected in tea products marketed as food. PURPOSE: The present study aimed at elucidating whether medicinal teas licensed or registered as medicinal products contain PAs as well. STUDY DESIGN: One hundred sixty-nine different commercially available medicinal teas, i.e. 19 nettle (Urtica dioica L.), 12 fennel (Foeniculum vulgare Mill.), 14 chamomile (Matricaria recutita L.), 11 melissa (Melissa officinalis L.) and 4 peppermint (Mentha piperita L.) teas as well as 109 tea mixtures were analyzed for the presence of 23 commercially available PAs. METHOD: LC/MS was used for the determination of the PAs RESULTS: In general, the total PA contents ranging 0-5668 µg/kg. Thirty percent of the tested single-ingredient tea products and 56.9% of the tested medicinal tea mixtures were found to contain PA concentrations above the limit of quantification (LOQ) of 10 µg/kg. In 11 medicinal teas PA contents >300 µg/kg dry herb were determined thus exceeding the recommended limit for PA intake by BfR. In addition three products of the investigated tea mixtures revealed extremely high PA contents of 4227, 5137, and 5668 µg/kg. Generally, single-ingredient tea products contained much less or even no detectable amounts of PAs when compared to the tea mixtures. PAs in the range between 13 and 1080 µg/kg were also detected in five analyzed aqueous herbal infusions of the medicinal tea mixture products with the highest PA content. Two out of the five investigated herbal infusions exceeded the recommended BfR limit for PA intake. CONCLUSION: This study demonstrates clearly that also medicinal teas licensed as medicinal products may partly contain high amounts of PAs exceeding current recommendations. For that reason manufacturers are advised to carry out more rigorous quality control tests devoted to the detection of PAs. This is very important to minimize PAs in medicinal teas accounting for possible additional exposure of the consumer to PAs from other food sources (e.g. honey).

Pharmacokinetic properties of MH84, a γ-secretase modulator with PPARγ agonistic activity.

Alzheimer's disease (AD) is the most common cause of dementia. Since no causative treatment is available, new therapeutic options are utmost needed. Several pirinixic acid derivatives, including MH84 (2-((4,6-bis(4-(trifluoromethyl)phenethoxy)pyrimidin-2-yl)thio)hexanoic acid), have shown promising in vitro results as γ-secretase modulators as well as PPARγ activators as potential pharmacological compounds against AD. Using a newly developed and validated sensitive LC-MS (APCI-qTOF mass analyzer) method, the pharmacokinetic and long-term accumulating properties as well as the blood-brain-barrier permeability of MH84 were evaluated in a preclinical animal study. MH84 was administered to mice by oral gavage with a dose of 12 mg/kg. Nine time points from 0.5 to 48 h with 6 animals per point were investigated. Additionally 6 animals were fed daily, for 21 days with an identical dose to determine possible long-term accumulation in plasma and brain tissue. The sample preparation was performed by a liquid-liquid extraction on Extrelut(®) columns whereas the LC separation was operated on a MulthoHigh 100 RP 18-5 µ column (125 × 4 mm) using an isocratic mobile phase of formic acid (0.1% (v/v))-methanol mixture (11:89 (v/v)) at a flow rate of 1 ml/min. The validation confirmed the new LC-MS method to be precise, accurate and reliable. After oral application, Cmax and Tmax of unmetabolized MH84 was determined to be 10.90 µg/ml and 3h in plasma. In brain tissue a constant level of 300 to maximum 320.64 ng/g was found after 1.5-6h. Daily gavage for 21 days did not lead to a long-term drug accumulation in the brain. The efficacy of the obtained MH84 levels needs to be investigated in further preclinical pharmacodynamic animal studies.

Advances in personalized medicine - medicinal chemistry and pharmacology of vemurafenib and ivacaftor.

Pharmacogenomics offers an entrance in the field of personalized medicine. This form of adapted therapy is going to be the future concerning the reduction of side effects and efficacy of the treatment of severe diseases. Vemurafenib and Ivacaftor are the first FDA approved drugs specially addressing mutated proteins. Both substances showed promising results in all clinical trials combined with relatively mild side effects by vemurafenib and placebo-like side effects by ivacaftor. The efficacy in addressing the specific mutation of each compound was confirmed in preclinical and clinical development.

Molecular pharmacological profile of a novel thiazolinone-based direct and selective 5-lipoxygenase inhibitor.

BACKGROUND AND PURPOSE: The potency of many 5-lipoxygenase (5-LOX) inhibitors depends on the cellular peroxide tone and the mechanism of 5-LOX enzyme activation. Therefore, new inhibitors that act regardless of the mode of enzyme activation need to be developed. Recently, we identified a novel class of thiazolinone-based compounds as potent 5-LOX inhibitors. Here, we present the molecular pharmacological profile of (Z)-5-(4-methoxybenzylidene)-2-(p-tolyl)-5H-thiazol-4-one, compound C06. EXPERIMENTAL APPROACH: Inhibition of 5-LOX product formation was determined in intact cells [polymorphonuclear leukocytes (PMNL), rat basophilic leukaemia-1, RAW264.7] and in cell-free assays [homogenates, 100, 000×g supernatant (S100), partially purified 5-LOX] applying different stimuli for 5-LOX activation. Inhibition of peroxisome proliferator-activated receptor (PPAR), cytosolic phospholipase A(2) (cPLA(2) ), 12-LOX, 15-LOX-1 and 15-LOX-2 as well as cyclooxygenase-2 (COX-2) were measured in vitro. KEY RESULTS: C06 induced non-cytotoxic, direct 5-LOX inhibition with IC(50) values about 0.66 µM (intact PMNL, PMNL homogenates) and approximately 0.3 µM (cell-free PMNL S100, partially purified 5-LOX). Action of C06 was independent of the stimulus used for 5-LOX activation and cellular redox tone and was selective for 5-LOX compared with other arachidonic acid binding proteins (PPAR, cPLA(2) , 12-LOX, 15-LOX-1, 15-LOX-2, COX-2). Experimental results suggest an allosteric binding distinct from the active site and the C2-like domain of 5-LOX. CONCLUSIONS AND IMPLICATIONS: C06 was identified as a potent selective direct 5-LOX inhibitor exhibiting a novel and unique mode of action, different from other established 5-LOX inhibitors. This thiazolinone may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer.

Isoquercitrin provides better bioavailability than quercetin: comparison of quercetin metabolites in body tissue and brain sections after six days administration of isoquercitrin and quercetin.

In the present study, over a period of 8 days 12 mg/kg/d quercetin aglycone and 18 mg/kg/d isoquercitrin were orally given to rats, respectively. Four hours after administration, plasma samples were taken as well as tissue samples of liver, lung, heart, kidney and the brain sections hippocampus, cerebellum, striatum, cortex and the remaining brain. A HPLC-FD method with in-line post-column complexation was employed to quantify the quercetin metabolites (QM) in plasma and tissues. Compared to the quercetin gavage the isoquercitrin gavage consistently produced higher levels of QM in tissues (double to five-fold) as well as in plasma (double to three-fold). In body tissues, the highest amounts of QM were observed in the lung. In brain tissue, the highest levels of QM were found in the cerebellum, while the striatum contained the lowest levels of QM. In conclusion, this study clearly demonstrates that orally given isoquercitrin leads to higher levels in plasma and in all investigated tissue than quercetin aglycone.

Glutathione peroxidase isoenzymes in human tumor cell lines.

A set of human tumor cell lines was characterized in terms of the GPx isoenzymes GPx1, -2, -3 and -4. Semiquantitative PCR was used to investigate the GPx mRNA transcripts and the GPx activity was determined photometrically. As a result of culturing under standard conditions, diverse distribution of GPx mRNA and basic GPx activity was found in the investigated cell lines. PCR results showed nearly ubiquitous existence of the isoenzymes GPx1 and GPx4. GPx2 mRNA transcript was only detected in the colonic cell line CaCo-2. After detection of the GPx3 mRNA transcripts in most of the tested cell lines, an ELISA was performed to investigate if the GPx3 protein is present as well. However, the GPx3 protein could not be detected. Glutathione peroxidases contain the amino acid selenocysteine in their active centre. Selenocysteine contains selenium instead of sulfur in cysteine. Therefore, the influence of selenium on GPx activity and GPx isoenzyme distribution was investigated. Cell culturing with additional selenium showed a clear elevation of GPx activity in Mono Mac 6 cells but no gain of mRNA transcripts or any change in the isoenzyme's distribution.

2-(4-(Biphenyl-4-ylamino)-6-chloropyrimidin-2-ylthio)octanoic acid (HZ52)--a novel type of 5-lipoxygenase inhibitor with favourable molecular pharmacology and efficacy in vivo.

BACKGROUND AND PURPOSE: 5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) representing a potential target for pharmacological intervention with inflammation and allergic disorders. Although many LT synthesis inhibitors are effective in simple in vitro test systems, they frequently fail in vivo due to lack of efficacy. Here, we attempted to assess the pharmacological potential of the previously identified 5-LO inhibitor 2-(4-(biphenyl-4-ylamino)-6-chloropyrimidin-2-ylthio)octanoic acid (HZ52). EXPERIMENTAL APPROACH: We evaluated the efficacy of HZ52 in vivo using carrageenan-induced pleurisy in rats and platelet-activating factor (PAF)-induced lethal shock in mice. We also characterized 5-LO inhibition by HZ52 at the cellular and molecular level in comparison with other types of 5-LO inhibitor, that is, BWA4C, ZM230487 and hyperforin. KEY RESULTS: HZ52, 1.5 mg·kg⁻¹ i.p., prevented carrageenan-induced pleurisy accompanied by reduced LTB(4) levels and protected mice (10 mg·kg⁻¹, i.p.) against PAF-induced shock. Detailed analysis in cell-based and cell-free assays revealed that inhibition of 5-LO by HZ52 (i) does not depend on radical scavenging properties and is reversible; (ii) is not impaired by an increased peroxide tone or by elevated substrate concentrations; and (iii) is little affected by the cell stimulus or by phospholipids, glycerides, membranes or Ca²âº. CONCLUSIONS AND IMPLICATIONS: HZ52 is a promising new type of 5-LO inhibitor with efficacy in vivo and with a favourable pharmacological profile. It possesses a unique 5-LO inhibitory mechanism different from classical 5-LO inhibitors and seemingly lacks the typical disadvantages of former classes of LT synthesis blockers.

A pirinixic acid derivative (LP105) inhibits murine 5-lipoxygenase activity and attenuates vascular remodelling in a murine model of aortic aneurysm.

BACKGROUND AND PURPOSE Arachidonic acid derivatives play a central role in inflammation processes. Arachidonic acid is metabolized by several enzymes, particularly cyclooxygenases (COX), 5-lipoxygenase (5-LOX) and microsomal prostaglandin E-synthase-1 (mPGES-1) to pro-inflammatory mediators. EXPERIMENTAL APPROACH We determined the effect of LP105, a pirinixic acid derivative which acts as inhibitor of 5-LOX, COX and mPGES-1, on aortic aneurysm development in mice and on 5-LOX activity in murine monocytes. KEY RESULTS In a monocyte cell line (RAW264.7), LP105 inhibited 5-LOX in whole cells (IC(50) : 1-3 µM) and in supernatants (IC(50) : ∼10 µM). Oral administration of LP105 to mice resulted in therapeutic tissue and plasma levels. Aortic aneurysms were induced in ApoE(-/-) mice by angiotensin II (AngII) and LP105 (5 mg·day(-1) per animal) was co-administered to a subgroup. Compared with animals receiving AngII alone, the LP105+AngII group showed a lower heart rate, a trend towards reduced heart to body weight ratio but similar hypertensive responses. AngII alone significantly increased aortic weight and diameter but co-treatment with LP105+AngII prevented these changes. LC/MS-MS studies revealed increased 15-hydroxytetraenoic acid (15-HETE) and 14,15-epoxyeicosatrienoic acid (14,15-EET) plasma levels in LP105-treated animals. In the murine kidney, mRNAs of EET-generating or metabolizing enzymes and of 5-LOX and 15-LOX were unaffected by LP105. LP105 also did not inhibit the EET-metabolizing soluble epoxide hydrolase. CONCLUSIONS AND IMPLICATIONS LP105 was a potent inhibitor of monocyte 5-LOX and reduced AngII-induced vascular remodelling in mice. A shift of arachidonic acid metabolism to the protective EET pathway may contribute to the beneficial effects of LP105.

Study of microbial contamination and dosing accuracy of oral dispensers.

BACKGROUND AND OBJECTIVE: The optimal administration of liquid medications requires accurate dose delivery. This is particularly important in the treatment of infants and children, as well as elderly people, who are more sensitive to dosage errors. Previous studies revealed significant dosage inaccuracies with measuring spoons. Oral syringes are therefore generally recommended instead. There is no data on the efficacy of standard cleaning techniques, and consequently on the degree of microbial contamination associated with the repeated use of oral syringes. This study aimed to investigate the level and types of microorganisms found in oral dispensers subjected to simulated in-use conditions. In addition, the dosing accuracy of the oral dispensers is compared with that of measuring spoons supplied and designed for use with specific medications. METHODS: Exadoral 5 mL oral dispensers from B. Braun Melsungen AG (Melsungen, Germany) were subjected to simulated in-use conditions and microbial assay. Six different liquid medications representing different substance classes were included. The test lasted 4-15 days with two to four doses withdrawn according to dosage recommendations. Dosing accuracy was assessed using six representative amoxicillin suspensions available on the German market after withdrawing 1.25, 2.5 and 5 mL that correspond to », ½ and full measuring spoons. RESULTS AND DISCUSSION: Low counts of Micrococcus luteus, Micrococcus lylae, Staphylococcus epidermidis, Staphylococcus chromogenes as well as Bacillus species and Candida lusitaniae may contaminate the interior surface of the oral dispenser, but the microbial count was below the accepted limit of microbial counts permissible for drinking water over the whole test period. Hence, oral dispensers may be considered safe, provided that cleaning procedures are followed exactly. Moreover, oral dispensers, although not specifically designed for the tested medication, showed much higher dosing accuracy in comparison with the specifically designed measuring spoons. CONCLUSION: The present study revealed that oral dispensers are accurate measuring devices for the safe administration of liquid medication. Pharmacists and physicians should encourage their patients to use oral dispensers routinely in practice.

Dosing accuracy of commonly used disposable insulin pens.

OBJECTIVE: The present study aimed to assess the dosing accuracy of commonly used disposable insulin pens including SoloStar (SR)*, FlexPen (FP) dagger, Next Generation FlexPen (NGFP) dagger, and KwikPen (KP) double dagger. It is the first comparative study covering the whole dosing range from 1 U to 60 U. It also covers the accuracy of SR at 80 U. RESEARCH DESIGN AND METHOD: A total of sixty insulin pens from two lots of each pen type were used. From each pen 1 U, 10 U, 30 U, 40 U, 60 U and 80 U were dispensed in random order. The 80 U dose was only evaluated for the SR as the other insulin pens do not deliver this dose in one injection. The actual doses were determined gravimetrically taking density of the different insulin preparations into account. The evaluation of dose accuracy was based on the regulations of the International Organization for Standardization (DIN EN ISO 11608-1:2000). RESULTS: All tested insulin pens met the requirements for accuracy with none of the single values at all dose levels being outside the defined range of the ISO recommendations (1 +/- 1 U, 10 +/- 1 U, 30 +/- 1.5 U, 40 +/- 2 U, 60 +/- 3 U and 80 +/- 4 U). For the investigated dosage levels the absolute average deviation of all insulin pens ranged between 0.09 and 0.81 U. CONCLUSION: The present study demonstrates an excellent dosing accuracy for all tested insulin pens, with no clinically relevant differences between the products.

St. John's wort flavonoids and their metabolites show antidepressant activity and accumulate in brain after multiple oral doses.

Over the last few years many data have been published suggesting a participation of quercetin flavonoids in the antidepressive effect of St. John's wort (SJW) extract. To elucidate these data more deeply we performed two animal behavioural studies examining the antidepressant effects of SJW extract, rutin and, in addition, the quercetin metabolite isorhamnetin. The substances were in all cases compared to imipramine using Porsolt's forced swimming test (FST) after oral gavage of the substances over a 9 day period. All three compounds were found to be effective, with isorhamnetin exhibiting the strongest effect. In addition to this pharmacological study, we carried out two pharmacokinetic studies to examine the CNS level time-curve of the quercetin flavonoids after a single oral dose of SJW extract (1600 mg/kg) and isoquercitrin (100 mg/kg), respectively, and to observe the cumulative effects after daily repeated oral doses of SJW extract over 8 days. After a single dose the maximal CNS levels for quercetin (340 ng/g) and isorhamnetin/tamarixetin (50 ng/g) were found at 4 h. With repeated doses the maximal cumulation for quercetin (367 ng/g) occurred after 5 days whilst isorhamnetin/tamarixetin (640 ng/g) did not reach its maximal cumulation level within the 8 day test period.

A rapid HPLC-UV method for the quantification of erythromycin in dermatological preparations.

According to the USP erythromycin determination in finished oral products as well as in some topical formulations is mainly carried out via microbiological assays. However, these assays are known for their long incubation periods, lack of precision and low sensitivity. In literature one HPLC method for the quantification of erythromycin in creams is described, which depends on electrochemical detection, but HPLC electrochemical detection has not emerged as popular choice in routine analysis. Furthermore two other HPLC-UV methods are described for the isolation of erythromycin from gels and creams involving tedious and time consuming extraction steps, the reason why they are not suited to be applied in routine analysis. This paper describes a new HPLC-UV method for the determination of erythromycin in creams, which implies a much easier extraction procedure than that cited in literature to date, based solely on the solubilization of erythromycin followed by freezing the cream matrix. Validation experiments confirmed the precision and accuracy of the method. Good linearity of the assay was found over the investigated concentration range of 70-130% (corresponding to 0.77-1.43 g of erythromycin A in 100 g cream base). The coefficient of correlation resulting from unweighted linear regression was 0.9998, allowing a one-point calibration in routine analysis. By the implementation of an internal standard in the quantification of erythromycin an improved precision could be achieved in routine analysis. This new analytical method yields cleaner extracts and allows a higher throughput, saving costs, solvents and time and can be thus recommended to all laboratories.

Screening of herbal extracts for activation of the human peroxisome proliferator-activated receptor.

The peroxisome proliferator-activated receptors play a pivotal role in metazoan lipid and glucose homeostasis. Synthetic activators of PPARalpha (fibrates) and PPARgamma (glitazones) are therefore widely used for treatment of dislipidemia and diabetes, respectively. There is growing evidence for herbal compounds to influence nuclear receptor signalling e.g. the PPARs. We recently reported carnosic acid and carnosol, both being diterpenes found in the labiate herbs sage and rosemary, to be activators of PPARgamma. The subsequent screening of a variety of ethanolic extracts, obtained from traditionally used herbs, for PPAR activation, led to an exceptionally high hit rate. Among 52 extracts nearly the half significantly activated PPARgamma and 14 activated PPARalpha in addition, whereas three of them were pan-PPAR activators, which also activated PPARdelta. The most active extracts, for which a concentration dependent effect could be shown, were the extracts of Alisma plantago aquatica (ze xie/european waterplantain), Catharanthus roseus (madagascar periwinkle), Acorus calamus (sweet calamus), Euphorbia balsamifera (balsam spurge), Jatropha curcas (barbados nut), Origanum majorana (marjoram), Zea mays (corn silk), Capsicum frutescens (chilli) and Urtica dioica (stinging nettle). The results of the present study provide a possible rationale for the traditional use of many herbs as antidiabetics.

Simultaneous determination of niacin, niacinamide and nicotinuric acid in human plasma.

A sensitive, specific, accurate, and reproducible HPLC/MS-method for the simultaneous quantitative determination of niacin (NA) and its main metabolites niacinamide (NAM) and nicotinuric acid (NUR) in human plasma using chinolin-3-carboxylic acid as an internal standard was developed and validated according to international guidelines for method validation. All analytes and the internal standard were separated from acidified plasma by solid phase extraction. Afterwards the extracted samples were analyzed by HPLC/MS in the positive electrospray ionization mode (ESI) and selected ion monitoring (SIM). The total run time was 7 min between injections. The assay had a lower limit of quantification of 50.0 ng/mL for each analyte using 1 mL of plasma. The calibration curves were linear in the measured range between 50.0 and 750 ng/mL plasma. The overall precision and accuracy for all concentrations of quality controls and standards was better than 15%. No indications were found for possible instabilities of niacin, niacinamide and nicotinuric acid in plasma at -20 degrees C, in the extraction solvent or after repeated thawing/freezing cycles. In stabilities were observed in whole blood and in plasma at room temperature. The recovery of the extraction method ranged from 86 to 89% for the three analytes.

Comparison of the synaptosomal uptake inhibition of serotonin by St John's wort products.

Although the number of prescriptions for psychotropic drugs has decreased in recent years, prescriptions for antidepressants are still increasing (Fritze 2002). Hypericum perforatum (St John's wort) is the main psychotherapeutic herbal medicinal product used for treatment of mild-to-moderate depression. The lipophilic constituent hyperforin (2-5% of the extract) demonstrated, similarly to chemical antidepressants, a significant effect on the synaptosomal uptake inhibition of several neurotransmitters in in-vitro assays. In Germany, St John's wort products are distributed via two different markets: products that are pharmacy restricted are only allowed to be distributed in pharmacies; traditionally used products, which do not claim to have a curative character, are allowed to be sold in supermarkets. Depending on the market wherein a St John's wort product is offered, it needs to fulfill the legal requirements regarding pharmaceutical quality, safety and efficacy. Our goal was to compare the quality of St John's wort products distributed in pharmacies with that of those available from supermarkets. Therefore, the quantity of the pharmaceutical active ingredients (the phloroglucinol derivate hyperforin, the flavonoids rutin, hyperoside, isoquercitrin, quercitrin and the biflavonoid biapigenin) was determined by high-performance liquid chromatography (HPLC). The naphthodianthrones hypericines and pseudohypericines were quantified by differential pulse polarography (DPP). The efficacy of the products was investigated by measuring their activity to inhibit serotonin (5-HT) uptake in-vitro using a radio ligand uptake assay. It could be demonstrated that the products were different not only in the concentration of pharmaceutically relevant ingredients but also in showing individual IC50 values (concentration producing half-maximal inhibition) in the serotonin reuptake assay (IC50 values between 3.07 and 17.9 microg extract mL(-1)). The results of our study confirm the assumption that the potency of St John's wort products in inhibiting the uptake of serotonin depends on the amount of hyperforin in their dosage forms. St John's wort products having greater hyperforin content and potency on synaptosomal serotonin uptake inhibition are restricted to be sold only in pharmacies.

Development of a high-performance-liquid-chromatographic method for the determination of biapigenin in biorelevant media.

A new precise, rapid and selective high-performance-liquid-chromatographic (HPLC) method has been developed to quantify biapigenin in St. John's Wort (SJW) preparations and to investigate its release characteristics in the dissolution test using both compendial and biorelevant media. Experiments were carried out on a LiChroCart 125-4, RP-18 (5 microm) column, using gradient elution at a flow rate of 1 ml/min. The binary mobile phase consisted of solvent A (acetic acid, 5:100, w:w) and B (a mixture of acetonitrile and methanol (3:1, v:v)). Detection was performed at a wavelength of 270 nm using a photodiodearray detector. The limit of detection was 0.05 microg/ml, the injection volume 20 microl. Five SJW preparations were chosen to determine the amount of biapigenin in the dosage form and to investigate their release characteristics. Best results in terms of release as well as discriminating the tested products were obtained, using fed state simulated intestinal fluid (FeSSIF), where over 80% of biapigenin was dissolved after 20 min comparing to 70% using simulated gastric fluid sine pepsin (SGF(sp)) as compendial medium. Experiments in fasted state simulated intestinal fluid (FaSSIF) show 80% release of biapigenin within 80 min.